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of propolis of native Brazilian stingless bees
Alexandra Christine Helena Frankland Sawaya, Ildenize Barbosa da Silva
Cunha, Maria Cristina Marcucci, Davi Said Aidar, Etelvina Conceição
Almeida Silva, Carlos Alfredo Lopes Carvalho, Marcos Nogueira Eberlin
To cite this version:
DOI: 10.1051/apido:2006058
Original article
Electrospray ionization mass spectrometry fingerprinting
of propolis of native Brazilian stingless bees*
Alexandra Christine Helena Frankland S
a, Ildenize Barbosa da Silva C
b,
Maria Cristina M
c, Davi Said A
d, Etelvina Conceição Almeida S
e,
Carlos Alfredo Lopes C
f, Marcos Nogueira E
aaThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, State University of Campinas, UNICAMP,
Campinas, SP 13083-970, Brazil
bSão Francisco University, USF, Bragança Paulista, SP, Brazil
cBandeirante University of São Paulo, UNIBAN, CEP 02071-013 São Paulo, SP, Brazil dCollege of Agricultural Sciences, Federal University of Amazonas, CEP 69077-000 Manaus, AM, Brazil
eETRON Apiary, km 13 Rodovia Ilhéus/Itabuna, (Ba 415) Ilhéus, BA, Brazil fFederal University of Recôncavo of Bahia (UFRB), 44380-000 Cruz das Almas, BA, Brazil
Received 11 April 2006 – Revised 23 June 2006 – Accepted 26 June 2006
Abstract – Stingless bees are found in many tropical and subtropical regions of the word. The knowledge of the composition of their propolis as well as the plants that are visited as sources of resins is therefore of prime importance. Here the negative ion mode electrospray ionization mass spectrometry [ESI(-)-MS] fingerprints of propolis from various species of native stingless bees from different regions in Brazil are compared to determine their composition patterns. The correlation among the propolis samples was inves-tigated via chemometric analysis.
ESI-MS fingerprint/ propolis / native stingless bees / Brazil
1. INTRODUCTION
Stingless bees (Hymenoptera, Apidae, Meliponini) are found in many tropical and subtropical regions of the word. They are the major visitors and native pollinators of flowering plants in the tropics, comprising a large group of small to medium sized bees with a level of social organization compa-rable to that of Apis mellifera bees (Heard, 1999). Native stingless bees are, however, less harmful to humans and domestic animals and resistant to the diseases and parasites of honeybees, and the propagation of their colonies contributes to the preservation of biodiversity (Aidar, 1996). Nevertheless, there Corresponding author: A.C.H.F. Sawaya,
[email protected] * Manuscript editor: Stan Schneider
is a poor level of domestication technology for most species of stingless bees (Heard, 1999). As their preferred nesting sites are the preformed cavities of live trees found mainly in primary forests, deforestation has decreased the density of these eusocial bees (Eltz et al., 2002). Therefore, information on the composition of the honey and propolis of these native bees, as well as the plants they visit as sources of pollen, nectar and resins, are of prime importance. Although studies have been performed on the overlap between native and introduced honey bees visiting flowers is several regions of Brazil (Wilms et al., 1996; Viana et al., 1997; Toledo et al., 2003), few studies have analyzed possible plant sources for the fabrication of propolis by stingless bees.
Bees collect exudates and resins from plants around their hives, adding wax to produce a
complex mixture of variable chemical compo-sition known as propolis. Bees use propolis to reinforce the combs and to keep the hive envi-ronment aseptic. A. mellifera propolis has been used by humans for many centuries for its mul-tiple pharmacological properties (Marcucci, 1995), hence its chemical composition and plant sources have been studied for nearly a century. In contrast, the chemical composition of stingless bee propolis has only recently be-gun to be studied. Bankova et al. (1999) stud-ied the essential oil content of propolis from three native stingless bees via gas chromatog-raphy mass spectrometry (GC/MS). Velikova et al. (2000a) studied the composition of ethanolic extracts of several samples of propo-lis from different species of native Brazilian stingless bees, concluding the chemical com-position of the propolis samples analyzed was heterogeneous. Velikova et al. (2000b) isolated three ent-kaurene diterpenoids from a sample of Melipona quadrifasciata (M.
quadrifasci-ata) propolis. Miorin et al. (2003) compared,
by high performance liquid chromatography with a diode array detector (HPLC-DAD), the chemical composition of the ethanolic extracts of several samples of Tetragonisca angustula and A. mellifera propolis from the states of Paraná and Minas Gerais in Brazil, conclud-ing that the composition T. angustula propo-lis differed from that of A. mellifera propopropo-lis from the same regions. Pereira et al. (2003) investigated by high temperature – high res-olution gas chromatography with mass spec-trometry (HT-HRGC/MS) the chemical com-position of the dichloromethane, acetone and methanol extracts of one sample of propolis of A. mellifera and one sample of T.
angus-tula from São Paulo, Brazil, concluding that
the less polar (dichloromethane) extracts were identical, but the other extracts showed sig-nificant differences in composition. Pino et al. (2006) compared by GC/MS the volatile con-stituents of one sample of A. mellifera propolis and one sample of Melipona beecheii propo-lis from Mexico, concluding that although the flora in the region was similar, the composi-tion of the two samples was different. In a recent study, we (Sawaya et al., 2006) used electrospray ionization mass spectrometry in the negative ion mode [ESI(-)-MS] to compare
ethanolic extracts of ten samples T. angustula propolis from the south, southeast and north-east of Brazil and compared their ESI(-)-MS fingerprints to the fingerprints of plant extracts from these regions, concluding that the almost constant composition of T. angustula propo-lis in Brazil results from the collection of sur-face exudates from Schinus terebenthifolius as the preferred plant source. This plant, known locally as “aroeira vermelha” can be found throughout South America.
Mass spectrometry has been used in con-junction with gas chromatography (GC/MS) for many years for the analysis of the main volatile and semi-volatile components of propolis from A. mellifera bees, although many components are not volatile enough for direct GC/MS analysis. HT-HRGC-MS was used to analyze the hexane and acetone ex-tracts of a sample of green Brazilian propolis (Pereira et al., 1998) while for the methano-lic extracts, prior derivatization was necessary (Pereira et al., 2000).
ESI-MS has revolutionized the way molecules are ionized and transferred to mass spectrometers, greatly expanding the applicability of MS to non-volatile, thermally unstable, heavy and/or polar molecules. Therefore ESI-MS and its tandem version ESI-MS/MS with direct infusion (no previous separation) has been applied to the analysis of a variety of complex natural mixtures such as those found in plant extracts (Mauri and Pieta, 2000; Möller et al., 2007), beer (Araújo et al., 2005), vegetable oils (Catharino et al., 2005; Wu et al., 2004) wine (Catharino et al., 2006; Cooper and Marshal, 2001), whisky (Möller et al., 2005), and even the most complex chemical mixture: petroleum (Hughey et al., 2002).
qualitative nature, one must bear in mind that it is not necessary to quantify all the com-pounds in a sample in order to characterize it. ESI(-)-MS fingerprinting focuses on the more polar and acidic components of the sample, which is of special importance for the study of propolis, as most of the pharmacologically active compounds so far identified in propolis are polar, frequently bearing acidic of pheno-lic sites (Marcucci et al., 1995). Therefore we have successfully applied ESI(-)-MS finger-printing to typify propolis from different geo-graphical regions, including the variable types of Brazilian propolis (Sawaya et al., 2004), and to compare propolis and plant extracts to determine the plant origins of the propo-lis samples (Marcucci et al., unpubl. data; Sawaya et al., 2006). These studies show, us-ing standardized conditions (see material and methods), that different types of propolis dis-play unique sets of polar and acid compo-nents hence that ESI(-)-MS fingerprinting can be used with confidence for propolis typifica-tion. In this study we compare the ESI(-)-MS fingerprints of ethanolic extracts of propolis of several species of native Brazilian stingless bees and some samples of A. mellifera propo-lis to determine patterns of composition for stingless bee propolis from different regions in Brazil. Furthermore, comparison with finger-prints of propolis samples with a known plant origin, permitted the identification of the plant origin of many of the propolis samples stud-ied herein. To evaluate the correlation among the propolis samples, chemometric principal component analysis (PCA) was applied to the ESI(-)-MS data.
2. MATERIALS AND METHODS 2.1. Propolis samples and extraction
procedure
Samples of propolis were provided by beekeep-ers from different regions in Brazil as summarized in Table I. All samples were frozen and ground prior to extraction. The samples were extracted by mac-eration for 7 days in a shaker, regulated at a speed of 100 opm and temperature of 30◦C, with 10 mL of absolute ethanol (Merck, Darmstadt, Germany) for every 3 g of crude propolis. The insoluble portion
was then separated by filtration, the filtrates kept in a freezer at –16◦C overnight and filtered again at this temperature to reduce the wax content of the ex-tracts. Solvent was then evaporated on a water bath at a temperature of 50◦C to obtain dry extracts of propolis.
2.2. General experimental procedures The dry propolis extracts were dissolved in a solution of 70% (v/v) chromatographic grade methanol (Tedia, Fairfield, OH, USA), 30% (v/v) deionized water and 0.1% ammonium hydrox-ide. The solutions of propolis were infused di-rectly into the ESI source by means of a syringe pump (Harvard Apparatus) at a flow rate of 10µL min−1. ESI(-)-MS and tandem ESI(-)-MS/MS were acquired using a hybrid resolution and high-accuracy (5µL/L) Micromass Q-TOF mass spec-trometer under the following conditions: capil-lary and cone voltages were set to −3000 V and −40 V, respectively, with a de-solvation temper-ature of 100 ◦C. For ESI(-)-MS/MS, the energy for the collision induced dissociations (CID) was optimized for each component. Diagnostic ions in the different propolis samples were identified by the comparison of their ESI(-)-MS/MS dissocia-tion patterns with compounds identified in previous studies (Sawaya et al., 2004, 2006; Marcucci et al., unpubl. data). Although fingerprints were acquired in the m/z 100−1000 range, no important ions were observed below m/z 200 or above m/z 650, there-fore ESI(-)-MS data is shown in the m/z 200−650 range.
2.3. Chemometric analysis of data Principal Component Analysis (PCA) was per-formed using the 2.60 version of Pirouette software from Infometrix, Woodinville,WA, USA. The mass spectra were expressed as the intensities of individ-ual [M - H]− ions (i.e. variables) of the ten most intense ions in the fingerprints of each sample. The data was preprocessed using auto scale and the PCA method was run.
3. RESULTS AND DISCUSSION
Table I. Species of bee and place of origin of samples of propolis from Brazil.
Sample Species Place of origin Region Ta1 Tetragonisca angustula Claudio, Minas Gerais Southeast Ta2 Tetragonisca angustula Florianópolis, Santa Catarina South Ta3 Tetragonisca angustula Cruz das Almas, Bahia Northeast Mq1 Melipona quadrifasciata Amazonas North Mq2 Melipona quadrifasciata Amazonas North Mq3 Melipona quadrifasciata Cruz das Almas, Bahia Northeast Mq4 Melipona quadrifasciata Ribeirão Preto, São Paulo Southeast Pr1 Plebeia remota Prudentopolis, Paraná South Pr2 Plebeia remota Prudentopolis, Paraná South
Pr3 Plebeia remota Paraná South
Pd Plebeia droryana Atibaia, São Paulo Southeast Pd3 Plebeia droryana Ubatuba, São Paulo Southeast
Ps1 Plebeia sp. Paraná South
Ps2 Plebeia sp. Itaparica, Bahia Northeast Ls Lestrimelitta spp. Paraná South Tc Tetragona clavipes Paraná South Nt Nannotrigona testaceicornis Minas Gerais Southeast Sb Scaptotrigona bipunctata Paraná South Ms1 Melipona scutellaris Bahia Northeast Ms2 Melipona scutellaris Cruz das Almas, Bahia Northeast Ms3 Melipona scutellaris Sauipe, Bahia Northeast Ms4 Melipona scutellaris Amazonas North Mf Melipona favosa Corumbá, Mato Grosso do Sul Midwest
Am1 Apis mellifera Paraná South
Am2 Apis mellifera Bahia Northeast Am3 Apis mellifera Cruz das Almas, Bahia Northeast
chemometric analysis. Figure 1 shows the PCA analysis of the ESI(-)-MS fingerprints of all the samples of propolis from stingless bees and A. mellifera collected in Brazil. Due to characteristic sets of polar and/or acid com-ponents, the samples are clearly divided into three main groups (Fig. 1A) mainly due to sev-eral diagnostic ESI(-)-MS ions circled in Fig-ure 1B. A detailed analysis of these ions al-lows us to indicate the main plant sources of these propolis samples (see below). To illus-trate, Figure 2 shows ESI(-)-MS fingerprints of typical samples of each group.
Group 1 is composed of the following nine propolis samples: three of T. angustula (from the Santa Catarina, Minas Gerais and Bahia); one of Nannotrigona testaceicornis from
Mi-nas Gerais; one of Plebeia sp. from Itaparica, Bahia; one of Plebeia droryana (P. droryana) from São Paulo; and the three remaining sam-ples were from Cruz das Almas, Bahia, that is one of A. mellifera, one of Melipona
scutel-laris (M. scutelscutel-laris) and one of M. quadri-fasciata. Figure 2 shows, as characteristic
examples of Group 1, a fingerprint of T.
an-gustula propolis from Minas Gerais (Fig. 2A)
Group 1 contain these same diagnostic ions (Fig. 1B), indicating that they derive their resins mainly from S. terebenthifolius, a tree 2–6 m high, belonging to the Anacardiaceae family, known in Brazil as “aroeira vermelha”. The leaves and fruit of S. terebenthifolius are popularly used for medicinal purposes and contain substances with known medici-nal properties (Jain et al., 1995; Schmourlo et al., 2005). References have been found to sev-eral species of native bees visiting this plant in the state of São Paulo (Ramalho et al., 1990) and in the south of Brazil (Wilms et al., 1997). Due to the wide geographic distribution of S. terebenhtifolius, it is hardly surprising that propolis samples from different regions (south, southeast and northeast of Brazil) use this plant source. The samples of T. angustula propolis analyzed in the present study were not the same ones analyzed in the previous pa-per (Sawaya et al., 2006), which reinforces our findings that S. terebenhtifolius is a major resin source for T. angustula.
Group 2 is also composed of 9 propolis samples: two of Plebeia remota (P. remota), one of Plebeia sp., one of Lestrimelitta sp., one of Tetragona clavipes, one of
Scaptotrig-ona bipunctata (S. bipunctata) and one of A. mellifera from Paraná; one of Melipona favosa from Mato Grosso do Sul and one of M. quadrifasciata from São Paulo. Figure 2D
shows a fingerprint of A. mellifera propolis from Paraná whereas Figure 2E shows that of
P. remota from Paraná, both characteristic
fin-gerprints of group 2. The ions of m/z 301, 315, 317, 319, 333, and 361 that are the most di-agnostic for these samples (Fig. 1B), which are characteristic of brown A. mellifera propo-lis from the south of Brazil. These ions are also found in resins of Araucaria pine trees and were characterized by comparison of their ESI-MS/MS (Marcucci et al., unpubl. data). Most of the samples of Group 2 come from the state of Paraná, where Araucaria trees are common and these resins are apparently the main source of propolis for bees in the south of Brazil. Furthermore, ions of m/z 253, 255 and 269 found in the fingerprints of both sam-ples of propolis (Fig. 2D−E) are also char-acteristic of brown propolis from the south of Brazil and were identified by ESI-MS/MS
as flavonoids commonly found in poplar type propolis in Europe (Sawaya et al., 2004). Al-though the plant source for these flavonoids in southern Brazil has not been determined yet it is noteworthy that both native (P. remota) and introduced (A. mellifera) bees take resins from this source. The sample of P. remota propolis from the south of Brazil (Fig. 2E) also con-tains compounds derived from S.
terebenthi-folius, as revealed by the detection of ions of m/z 453 and 471 in the ESI(-)-MS fingerprint,
albeit with low intensity. Due to matrix sup-pression, one cannot affirm that the intensity of an ion is proportional to its concentration.
Two samples of propolis (P. droryana from São Paulo and P. remota from Paraná) were placed between Groups 1 and 2 in the PCA analysis (Fig. 1A), which indicates that both
S. terebenthifolius and Araucaria were used
as plant sources. Figure 2C shows the finger-print of such a sample (P. droryana propolis from São Paulo) where the ions of m/z 301 and 319 (characteristic of Araucaria) as well as m/z 373, 401, 455 and 471 (characteristic of S. terebenthifolius) are observed.
Group 3 is composed of the following six propolis samples: two of M. scutellaris from Bahia and one of M. scutellaris from zonas; two of M. quadrifasciata from Ama-zonas and one of A. mellifera from Bahia. By far the major diagnostic ion responsible for grouping these samples is that of m/z 271 (Fig. 1B), and this ion is very characteristic for the ruby redA.mellifera propolis from the northeast of Brazil (Sawaya et al., 2004). Al-though the plant source of this type of propo-lis has not been determined yet, it is appar-ently a plant source for propolis of several species of bees in the tropical region of Brazil. Figure 2F shows the ESI(-)-MS fingerprint of ruby red A. mellifera propolis from Bahia. The ion of m/z 601 is characteristic of ruby red propolis from the coastal regions of the state of Bahia and was observed with less intensity in several samples of stingless bee propolis from this state (data not shown). Fig-ure 2G shows a fingerprint of M.
quadrifasci-ata propolis from Amazonas where the ion of m/z 271 is the most intense, but less intense
ions which are characteristic of S.
Figure 3. Map of Brazil indicating the geographic origin of the propolis samples. Sample names abbreviated as in Table I.
observed. Both fingerprints are characteristic of Group 3 samples.
Most of ESI(-)-MS fingerprints of the sam-ples of propolis from native Brazilian sting-less bees contained ions characteristic of S.
terebenthifolius, whereas only three of the
samples analyzed contained no trace of these ions (two samples of M. scutellaris from Bahia and Amazonas, and one of S. bipunctata from Paraná). This finding is consistent with reports in which numerous stingless bees were found visiting S. terebenthifolius flowers (Ramalho et al., 1990; Wilms et al., 1997). These results indicate that S. terebenthifolius is an impor-tant plant source for propolis of native Brazil-ian stingless bees. In regions where other plant sources are present, stingless bees can adapt and use different plants (such as Araucaria in the southern regions of Brazil).
A map of Brazil indicating the states and regions in which the samples of propolis were collected can be seen in Figure 3. Most of the samples were collected in tropical regions, with a wide variety of vegetation. The sam-ples collected in the south of Brazil, states of Paraná and Santa Catarina, show the influence
of the vegetation, with Araucaria trees being an important plant source for these samples. In the southeast of Brazil, A. mellifera bees use
Baccharis dracunculifoia, as the main plant
source for propolis, resulting in the world fa-mous green Brazilian propolis (Bankova et al., 1999; Marcucci et al., unpubl. data). Diag-nostic ions of the prenylated phenolic com-pounds found in green A. mellifera propolis (Sawaya et al., 2004) were not found in any of the fingerprints of native stingless bees, in spite of containing both acid and phenolic sites and ionizing well in the negative mode. There-fore we observed that, even in the southeast of Brazil where this shrub is common, native bees do not use B. dracunculifoia as a plant source for their propolis.
4. CONCLUSIONS
469 and 471, are the most intense in the fin-gerprints of all the samples of T. angustula and N. testaceicornis analyzed. These ions are also present in all the samples of propolis from the species of Plebeia analyzed, and in sev-eral samples of the other stingless bee species (eg. Fig. 2F). Stingless bees can however use many of the same plant sources as A. mellifera; in the fingerprints of samples of stingless bee propolis from the south of Brazil, the charac-teristic ions of Araucaria resins (m/z 301, 319) and some flavonoids (m/z 253, 255 and 269) are observed that are common in A. mellifera propolis from this area. What is more surpris-ing, is that sometimes native bees seem to sim-ply ignore a potential plant source used by A.
mellifera. For example, none of the
character-istic ions of green A. mellifera propolis are ob-served in the fingerprints of any of the samples of stingless bee propolis from the southeast of Brazil, indicating that Baccharis
dracunculi-folia was not used as a source of resins by any
of the stingless bees studied.
ACKNOWLEDGEMENTS
This work has been supported by the São Paulo State Research Foundation (FAPESP) and the Brazilian National Research Council (CNPq). Spectrométrie de masse par ionisation avec élec-tronébulisation, une méthode par fingerprint pour caractériser la propolis des abeilles sans aiguillon indigènes du Brésil.
propolis/ spectrométrie de masse par ionization avec électronébulisation/ abeille sans aiguillon / Apidae/ Meliponini / Brésil / Schinus thereben-tifolius
Zusammenfassung – Elektrospray-Ionisations-Massenspektrometrie, eine Methode zur Fin-gerabdruckanalyse von Propolis brasilianischer Stachelloser Bienen. Stachellose Bienen kommen in vielen tropischen und subtropischen Regionen der Welt vor und sind in diesen Regionen wichti-ge Bestäuber. Nichtsdestotrotz ist die Haltungstech-nologie für die meisten Arten Stachelloser Bienen noch auf einem relativ niedrigen Niveau. Obwohl für Brasilien bereits verschiedene Studien zur Ni-schenüberlappung von einheimischen Bienen mit den eingeführten Honigbienen vorliegen, gibt es nur wenig Informationen zu Pflanzen, die von Sta-chellosen Bienen als Harzquelle für die Herstellung
von Propolis genutzt werden. Propolisproben, die von Imkern in verschiedenen Regionen Brasiliens gewonnen wurden (zusammengestellt in Tab. 1), wurden eingefroren und für die Extraktion zer-mahlen. Die mazerierten Proben wurden auf einem Schüttler während sieben Tagen in Alkohol extra-hiert und anschliessend im negativen Ionenmodus per Elektrospray-Ionisations-Massenspektrometrie [ESI(-)-MS] in einem Q-TOF-Massenspektrometer (Micromass) analysiert. Mittels einer chemome-trischen Hauptkomponentenanalyse (PCA) wur-den statistisch signifikante Korrelationen in diesen Fingerabdrucksanalysen der Propolisproben aufge-deckt. Abbildung 1 zeigt die PCA-Ergebnisse der ESI(-)-MS Fingerabdrücke von Propolisproben Sta-chelloser Bienen und von Honigbienen. Die Proben teilen sich anhand ihrer charakteristischen Ionen klar in drei Gruppen auf (Abb. 1A, B). Abbildung 2 zeigt ESI(-)-MS-Spektren typischer Proben aus je-der dieser Gruppen. Gruppe 1 besteht aus neun Pro-polisproben, für die die Ionen m/z 371, 373, 401, 453, 455, 469 und 471 für die Gruppierung ver-antwortlich sind. Diese Ionen sind charakteristisch für Tetragonisca angustula Propolis, die diese Bie-nen in ganz Brasilien überwiegend von Schinus ter-ebenthifolius sammeln. Gruppe 2 besteht ebenfalls aus neun Propolisproben, mit den Gruppierungsio-nen m/z 301, 315, 317, 319, 333, and 361. Diese sind für die braune A. mellifera Propolis charakte-ristisch, die vor allem aus Südbrasilien stammt und in der die Bienen Harze der Araucaria Tanne verar-beiten. Zwei Propolisproben (P. droryana aus São Paulo und P. remota aus Paraná) lagen zwischen diesen beiden Gruppen (Abb. 1A), was darauf hin-weist, dass diese Bienen sowohl S. terebenthifolius als auch Araucaria Harze sammelten. Gruppe 3 be-steht aus sechs Propolisproben, für die das Haupti-on (m/z 271) für die Gruppierung verantwortlich zeigt. Dieses ist charakteristisch für rote Robinien-propolis von A. mellifera aus dem Nordosten Brasi-liens. Die meisten Fingerabdrücke von Propolispro-ben der einheimischen Stachellosen Bienen zeig-ten die für S. terebenthifolius charakteristischen Io-nen. Diese in ganz Brasilien vorkommende Pflan-ze enthält medizinisch wirksame SubstanPflan-zen und wird häufig von Stachellosen Bienen besucht. Un-sere Ergebnisse zeigen, dass S. terebenthifolius ei-ne wichtige Quelle für die Propolisgewinnung dar-stellt, dass Stachellose Bienen aber auch andere Pflanzen, insbesondere Aurakarien nutzen können. ESI-MS Fingerabdruck/ Propolis / Stachellose Bienen/ Brazil
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